Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these references is incorporated herein as though set forth in full.
Direct somatic cell nuclear injection into a recipient “enucleated” oocyte, which is subsequently cultured under conditions conducive to the formation of blastocysts to be transplanted into a surrogate mother, allows for the production of individuals genetically identical to the donor of the cell (i.e. clones). The production of these clones provides a means for: i) reproduction of a species that cannot reproduce effectively on its own (e.g. endangered species); ii) the production of animals with certain desirable or commercially valuable characteristics (e.g. better performance of dairy farm species); iii) deriving individual specific stem cells from the blastocyst (e.g. therapeutic cloning); and iv) treating infertility given the ability to turn embryonic stem cells into germ cells and ultimately mature eggs (Hubner, K. et al. (2003) Science). Additionally, the nucleus donor cells may be manipulated prior to nuclear transfer, so as to add, subtract, or amend any characteristic that will then be incorporated into the germline of the cloned animal and be transmitted to subsequent generations.
The currently employed methods for cloning animals are very inefficient and often fail to provide reproducible results. The two main procedures used for cloning mammals are the Roslin method and the Honolulu method. These procedures were named after the generation of Dolly the sheep at the Roslin Institute in Scotland in 1996 (Campbell, K. H. et al. (1996) Nature 380:64-66) and of Cumulina the mouse at the University of Hawaii in Honolulu in 1998 (Wakayama, T. et al. (1998) Nature 394:369-374). In contrast to the cloning of farm species based on the Roslin method, the technique used in the cloning of mice by the Honolulu method has remained elusive.
The ability to clone laboratory animals, such as mice, is highly desirable for several reasons. Clones can provide material for therapeutic cloning. The ability to produce embryonic stem (ES) cells has only been established in mice and humans. The mouse is the only species where an example of therapeutic cloning has been accomplished and where ES cells have been differentiated into mature oocytes, though the employed techniques may be employable in other species including humans (Rideout, W. M. et al. (2002) Cell 109:17-27; Hubner, K. et al. (2003) Science). Moreover, the handling and costs associated with a mouse model are practical and the genome is well studied. Additionally, the gestation period of the mouse is only 3 weeks as compared to 9 and 4 months for bovine and ovine species, respectively.
Cloned animals have exhibited a similar set of defects regardless of species. Specifically, dysregulation of gene expression during embryogenesis, placentomegaly during gestation, respiratory failure at birth, and conditions in the adult such as obesity, immunodeficiency and seizure are common. No corrective procedures to eliminate such defects have yet been developed that do not involve the passage through the germline (Tamashiro, K. L. et al. (2002) Nat. Med. 8:262-267) or the derivation of ES cells (Rideout, W. M. et al. supra). While these interventions can benefit the next generation or the adult, they are completely ineffective for correcting defects during prenatal stages. It is at the prenatal stages, however, where the greatest amount of attrition in clones occurs. Additionally, current procedures seldom result in the delivery of more than one clone per gestation in those species where this is naturally the case (e.g. mouse, pig) and call for extraordinary perinatal care. Therefore, improved methods which minimize this attrition during the prenatal stages would be most advantageous.
All of the defects cited above are related, directly or indirectly, to alterations that occur in the expression of the genetic material of the donor nucleus upon transplantation into the oocyte. There is a consensus that “reprogramming” of the donor genetic material begins soon after transplantation, but this “reprogrammed” nucleus is seldom equivalent to the state of a zygotic nucleus (i.e. reprogramming is either incomplete or faulty). Indeed, an analysis of global gene expression by cDNA microarray technology shows that the expression of a number of genes is consistently biased due to the general cloning procedure employed while other genes are abnormally expressed in relation to the type of donor nucleus (Humpherys, D. et al. (2002) Proc. Natl. Acad. Sci. 99:12889-12894). The level of reprogramming obtained likely relates to the subsequent phenotype of the clone animal. It is unclear, however, to what extent gene expression defects at early stages practically contribute to subsequent postimplantation impairment or demise of clones. A viable approach to gene expression analysis (e.g. a GFP-tagged transgene reporter) may allow the determination of the effects of certain gene expression defects (Boiani, M. et al. (2002) Genes Dev. 6:1209-1219).
The expression of the POU-domain transcription factor Oct4, which is expressed in pluripotent cells and is evolutionarily conserved in humans, bovine, and mice (Nordhoff, V. et al. (2001) Mamm. Genome 12:309-317), is required at the morula stage to prepare for the first cell lineage decision (Nichols, J. et al. (1998) Cell 95:379-391). In normal blastocysts, Oct4 expression is maintained only in the inner cell mass and downregulated in the trophectoderm. It has been previously demonstrated that the distribution and level of Oct4 is abnormal in 2 out of every 3 somatic cell derived mouse clones at the blastocyst stage and this ratio correlates well with the inability to form ES cells (Boiani, M. et al. (2002) Genes Dev. 6:1209-1219). Notably, gap junction mediated signaling has been shown to induce Oct4 expression in epithelial-like cells placed in contact with embryo blastomeres (Burnside, A. S. and P. Collas (2002) Eur. J. Cell. Biol. 81:585-591). This indicates that alternative routes for reprogramming may exist in addition to or in association with the direct exposure of a donor nucleus to the ooplasm.
Typically, much less than 50% of clone mouse embryos produced by current techniques attain a level of reprogramming compatible with blastocyst formation. Additionally, less than 5% of clone mouse embryos attain a level of reprogramming compatible with postimplantation development and birth. It is therefore highly desirable that improved methods be developed to increase viable mouse clone yields.